...my live-dead staining. (B) Changing the y-axis from SSC to FSC can alter the positioning and shape of ...After several steps of tissue processing prior to running my flow analysis, I can however clearly identify...’s always good to ask around for advice before running your analysis. In this case, I and other lab members...and other larger cells. Note, we don’t mind the warning message at the top, as our cells of interest do...ones (Figure 3). A more “liberal” gate in the beginning should not have a great impact on your analysis...gating pipeline, it might be worth going back and loosening the FSC/SSC gate.   Single-cell gating Once you...duplicates can’t be reliably analyzed on their staining signals. For this, you’ll need to gate based on...

...Restriction Cloning Get Tips on Analyzing DNA Sequencing Results Check out Plasmid Cloning by PCR Resources...applications range from confirming the results of cloning or knockout experiments to forensics and clinical...including easier automation, higher throughput screening, and a lower requirement for the amount of DNA...unknown sequence with restriction enzymes and then running the products on an agarose gel allows a researcher...Labs still keep restriction enzymes on hand for cloning and analytical digests. A researcher can test the...dideoxy sequencing, was a major breakthrough for determining the order of nucleotides in a string of DNA (...sequencing, such as confirming the results of a cloning experiment, require only one or a few sequencing...

... fall under informal learning; however, many others partner with formal learning to engage students at...environment such as the classroom or a defined learning program. Learners cannot (theoretically, at least...are interested in breeding dogs who also enjoy listening to podcasts A series of Tweets breaking down...caregiver communities  Table 1: Examples of defining your audience for science communication activities...activities  Many people are concerned that by defining a specific audience, they’ll exclude others from accessing... to you and a few other people on your team?)  Defining your audience allows you to create common ground...re ready to get into the more exciting parts - learning more about science communication itself! See you...

...predictable cut site) to develop the first plasmid “cloning” experiment. Dr. Cohen and colleagues treated a...proved successful, pSC101 became the first plasmid cloning vector and molecular biology was never the same...mammalian) species were cloned into plasmids and new cloning vectors such as pBR322, pACYC, and pUC were developed...copy number vectors that could be used in these cloning experiments. Although plasmids started as a somewhat...and recombination, replication and topology, and cloning and gene expression. Just released: Addgene'...eBook include: new chapters on genome engineering, cloning, and plasmid tags, addtional content added to the... Learn more about the history of plasmids and cloning: CSHL Meeting: Plasmids: History & Biology The ...

...validated. The creativity in employing a variety of cloning techniques and troubleshooting methodologies drew...colony PCR.   Fig. 1: (A) Schematic of cloning, expression and validation pipeline. (B) Schematic...the research community.    Validation For most cloning projects, the next step would be to validate your...which was observed for the hybridoma mAb.  While screening by IF-ICC worked well for identification of the...performing a BciVI digest of the PCR products prior to cloning, which in most cases left the functional light ...Approximately 70% of mouse mAbs are IgG1 subclass (Manning et al. 2012), which limits the use of subclass ...collection References Andrews NP, Boeckman JX, Manning CF, Nguyen JT, Bechtold H, Dumitras C, Gong B, ...

...Restriction enzyme cloning is the workhorse of molecular cloning; however, one of its biggest limitations...sequence. This means cloning occurs in vivo, as compared to restriction enzyme cloning where the genetic ...mutations and will have more unintended changes to their genomes. 2) Designing ssDNA oligos that avoid MMR... is an alternative method that can be used for cloning or genome engineering and is based on homologous...that differ from traditional restriction enzyme cloning will be explained in greater detail. Substrate... and can be increased to as high as 25-50% by designing oligos that avoid activating the methyl -directed...

... but educators face significant challenges in designing suitable projects. In the O'Connor lab, we recognized... about the evolution of the genes involved in methionine synthesis (5). The budding yeast, Saccharomyces...-annotated deletion strains is available (6). Methionine synthesis in S. cerevisiae (Figure 1) occurs ... transformed strain to grow in the absence of methionine (i.e. the plasmid complements the deletion). ...enable multiple student experiments We used TOPO cloning to insert the coding sequences for ten different...plasmid is controlled by the GAL1 promoter. The cloning procedure adds both V5 and His6 epitope tags to...strains on media that lack uracil, but contain methionine. (This allows cells carrying nonfunctional as...

...check out the distance learning courses at the National Council for the Training of Journalists (NCTJ) ...guided tours to show people where the lab magic is happening. 3) Volunteer – Step into science education Do...Learn on the job Nothing prepares you better than learning on the job. Try to get an internship at a magazine...internships to cover their annual meetings. 8) Training – Get a formal education Some people get into ...engagement for institutions and the Royal Society gives training in media skills, writing about your research and...moment, I got active with it, new doors started opening up. My current job as a Marie Curie Fellow is a... Additional Resources on Addgene.org Find Job Openings at Addgene ...

...allencell.org and the Allen Institute website at alleninstitute.org. A classic challenge in cell biology is making...will add more to the collection later this year. Designing the donor plasmid Figure 2: Donor plasmid...make a precise cut in the genome near either the beginning or end of the gene of interest —in our case in... without outside influence (non-homologous end joining, or NHEJ), in approximately one percent of cases... process. By flooding the cell with a plasmid containing regions of high homology to the gene with the... relying instead on microhomology-mediated end-joining (MMEJ). To find out more about gene edited hiPS...allencell.org and the Allen Institute website at alleninstitute.org. References 1. Roberts, Brock, et al. "Systematic...

...back in 2007, which function as centres for peer learning and knowledge sharing, in the form of workshops...maintains a list of active spaces near you.   Learning about C. elegans with the OpenWorm Project One...variety of aspects from the molecular biology to learning how to grow and care for the worms. As an example...relies upon biologists and computer scientists learning and communicating with one another.      Currently...declared our makerspace an educational institution meaning that we can obtain known mutants at $7.00 a strain...optimizing the health of domesticated cats and learning about feline microbiomes. One of the aspects of...the kittybiome project, but the primary goal is learning in this case.   Funding Makerspaces One of the...

...requirements and expression limitations may be too constraining for some experiments, particularly in vivo ones...mice, AAV is almost entirely non-immunogenic — meaning when conducting in vivo CRISPR screens, AAV is ... This vastly increases the possibilities when designing screens. Each serotype has its own tropism, or... If you don’t feel like doubling the amount of cloning, another option is to use variants of Cas9, like...immunotherapy in glioblastoma, and generate programmable screening platforms for mouse tissues. Your AAVancement ..., Palop, J. J., & Kampmann, M. (2023). CRISPR screening by AAV episome-sequencing (CrAAVe-seq) is a highly...highly scalable cell type-specificin vivoscreening platform. bioRxiv (Cold Spring Harbor Laboratory). ...

...that many researchers feel that they lack formal training in a variety of transferable skills. At Addgene...about management skills during your scientific training. I never thought about management skills before...grad school. Both were hard-working scientists-in-training who got the job done, but I struggled with what... he graduated. Years later I attended a formal training for new managers and I had a “Eureka” moment while... people! More specifically, they had different learning styles. One was a “Doer” and the other was a “...different levels of experience), find out what their learning styles are and capitalize on them for the benefit...it so shouldn't it be faster to do it yourself? Training certainly takes time - so plan accordingly -  ...

...Addgene: Tell me about the Golden Gate Modular Cloning (MoClo) Kit. What does it include? Patron: Sainsbury...already use. It makes the switch to Golden Gate Cloning easier because individual labs will not need to...make it easier for people to use the Golden Gate cloning system especially with plants in mind. When it’...There is no PCR, no gel purification; the whole cloning process is so simplistic once you have your parts... 30 constructs. They’ve never done any kind of cloning and they don’t have any prejudice about it. They... Sometimes it’s a good way to find out what’s happening in the office next to you. There is an active ... biology.  References: A Golden Gate Modular Cloning Toolbox for Plants. Carola Engler, Mark Youles,...

...that we have the beginning of control around the central dogma. We have the beginnings of computational...program created as part of President Barack Obama's Winning the Future Initiative. Endy's team made news in... - enable? Endy: The BIOFAB collection is the beginnings of what we think of as an operating system for...expression levels. The BIOFAB project is seeding the beginnings of an operating system for programming life, ...'ve described the pieces in these kits as the beginnings of an operating system or language for programming...percent of the U.S. gross domestic product – equal to mining. There is an absolutely incredible hidden story...genetic elements that allow you to begin to imagine designing 10-gene systems with quantitative precision. There...

...besides HR - Non-homologous end-joining (NHEJ) and alternative end joining (alt-EJ). Inhibitors to essential...harnessing the homology-directed repair pathway, designing the best donor DNA, and avoiding common mishaps...’s no surprise then that these edits take more planning and finesse then their knock-out counterparts....strand break by homologous recombination.    Designing donors for HDR The basis of the HDR pathway relies...used as well. Below are some considerations for designing donor DNA for successful HDR events. CRISPR cut...reading up on validating genome edits to find screening strategies for your experiment. Also learn about...Featured Topic Page Learn about Non-Homologous End Joining Learn about Base Editing Read 3 Tips to Improve...

...You want to halt degradative processes while maintaining the structure of your samples, the relationships... it is great for preserving your samples and maintaining cellular and tissue morphology. Formaldehyde ...detergents, such as Triton X-100, Tween-20, or saponin. Each detergent has different chemical properties... and membrane integrity (Fig. 2). For example, saponin interacts with cholesterol in cell membranes and...) Permeabilizing with the selective detergent, saponin, keeps membrane-associated proteins in place, while...fixing with formaldehyde and permeabilizing with saponin. However, if you are not worried about washing ...https://doi.org/10.1177/33.8.3894499    Im K, Mareninov S, Diaz MFP, Yong WH (2018) An Introduction to...

...introduced through non-homologous end joining (NHEJ). Designing an RNA editor The Zhang lab envisioned...would convert adenine to inosine, which the translational machinery treats like guanine. This RNA editor...gRNA spacer, as ADAR will preferentially edit an adenine if the template has a cytosine mismatch at that... natural cytidine deaminases, they evolved the adenine deaminase ADAR2DD to deaminate cytidine and used...-U edits, the authors note that it retains its adenine deaminase ability. The authors then tested the ...around the targeted nucleotide. Introduction of guanine-mismatches in the guide across from these off-target...

...For these reasons, we are in the process of transitioning our titering method from qPCR to ddPCR. Droplet...ddPCR) does not require a standard or a reference, meaning saving on reagents (and time)! AAV titering using...droplet digital PCR Droplet digital PCR involves partitioning a PCR reaction mixture into approximately 20,000...ingredients for amplification of the target DNA. This partitioning reduces the number of PCR inhibitors per reaction...replicates are built into the technology. One well (containing thousands of droplets) can be sufficient to capture...dilutions, they are transferred to a new plate containing the mastermix which includes primers and a ddPCR...small channels to create a water-in-oil emulsion containing approximately 20,000 droplets. Each droplet contains...

...another genetic disease more prevalent or life-threatening (cystic fibrosis, hemophilia, muscular dystrophy... years ago when completing her PhD and medical training in Ophthalmology. Gene therapy was in its infancy...Importantly, it is an immune privileged compartment, meaning that it can tolerate the presence of antigens (... Congenital Amaurosis (LCA). Shortly after the cloning of the RPE65 gene, a naturally occurring animal...technology: engineering of new capsid variants, designing expression cassettes to regulate and optimize ...reports suggest that the first clinical study combining these tools may start as early as next year. Twenty... areas of science. This is hopefully only the beginning of a gene therapy revolution and the development...

...Enable Genome-Scale, Knockout Screening Read about Genome-Wide Screening Using CRISPR/Cas9 Additional...these plasmids, 293T cells released transgene-containing lentiviral particles into the media, which could...eliminated through the addition of a chimeric 5’ LTR containing a Tat-independent promoter. Although safer, this...both for targeted modification and genome-wide screening.   Lentiviral vectors in the clinic Since lentiviral...HIV-1 were transduced with a lentiviral vector containing an antisense sequence against the HIV-1 envelope...lentiviral plasmid and biosafety guides before beginning your experiment. Let us know how you use lentivirus... vectors and genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nat Methods. 2014...